This application relates to methods of treating biological materials, such as blood products, with a nucleophilic compound to quench reactive electrophilic compounds in the material.
The transmission of disease by blood products and other biological materials remains a serious health problem. While significant advances in blood donor screening and blood testing have occurred, viruses such as hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) may escape detection in blood products during testing due to low levels of virus or viral antibodies. In addition to the viral hazard, there are currently no licensed tests to screen for the presence of bacteria or protozoans in blood intended for use in transfusions. The risk also exists that a hitherto unknown pathogen may become prevalent in the blood supply and present a threat of disease transmission, as in fact occurred before the recognition of the risk of HIV transmission via blood transfusions.
Exposure of laboratory workers to blood or other body fluids also presents a health hazard. As recently as 1989, the Centers for Disease Control estimated that twelve thousand health-care workers whose jobs involve exposure to blood are infected with hepatitis B virus each year. xe2x80x9cGuidelines for Prevention of Transmission of Human Immunodeficiency Virus and Hepatitis B Virus to Health-Care and Public-Safety Workers,xe2x80x9d Morbidity and Mortality Weekly Report, vol. 38, no. S-6, June 1989. This statistic illustrates the need for methods to inactivate pathogens in biological materials.
Chemical agents have been introduced into blood or blood plasma to inactivate pathogens prior to clinical use of the blood product. Methods and compositions for photochemical inactivation of pathogens have been described. U.S. Pat. Nos. 5,587,490 and 5,418,130 describe substituted psoralens used for photochemically inactivating viral or bacterial contaminants in body fluids. Phenothiazines such as methylene blue have been shown to inactivate pathogens in blood products upon illumination. Wagner et al., Transfusion, 33:30-36 (1993). U.S. Pat. No. 5,637,451 describes a method for inactivating viruses in a red blood cell containing material by adding a phthalocyanine compound and irradiating the material.
The disadvantage of photochemical methods for pathogen inactivation is that the reactive free radicals and oxygen species that are generated also can cause damage to blood products and compromise their suitability for their intended use. Free radical quenchers have been used in the photochemical methods for pathogen inactivation, to reduce and minimize free radical damage which occurs during the photochemical reaction, as described in U.S. Pat. Nos. 4,727,027, 5,587,490, 5,418,130, 5,232,844, 5,658,722 and 5,637,451, and International Patent Application WO 97/16966.
Compounds have been developed for pathogen inactivation which do not require photoactivation. These compounds are typically electrophiles that react with pathogens. For example, U.S. Pat. No. 5,055,485 describes the inactivation of viruses in cell and protein containing compositions using aryl diol epoxides. Other compounds generate electrophiles in situ. LoGrippo et al. evaluated the use of nitrogen mustard, CH3N(CH2CH2Cl)2, for viral inactivation. LoGrippo et al., Proceedings of the Sixth Congress of the International Society of Blood Transfusion, Bibliotheca Haematologica (Hollander, ed.), 1958, pp. 225-230. More significantly, U.S. Pat. Nos. 5,691,132 and 5,559,250, the disclosures of which are incorporated herein by reference, describe the use of N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine (xe2x80x9cquinacrine mustardxe2x80x9d) and 5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralen for pathogen inactivation in blood, blood products, and a variety of samples of biological origin. Pathogen inactivating compounds which include an aziridine covalently attached to a polyamine moiety also have been used, as described in Budowsky et al., Vaccine Research 5:29-39 (1996).
Ideally, addition of the pathogen inactivating compounds that inactivate by electrophilic reactions or intermediates to the blood product or other biological sample would inactivate pathogens without causing any undesirable modifications to the sample. The pathogen inactivating compounds react with pathogens by an electrophilic process, and do not require photoactivation. Therefore reactive oxygen or free radical species are not produced, and oxidative damage is not a concern. Other undesirable side reactions, however, may occur. For example, electrophilic reactions with different biological materials, including proteins may occur. These side reactions can potentially compromise use of the biological sample for its intended purpose.
Thus, there is a need for methods to prevent unwanted electrophilic side reactions of pathogen inactivating compounds that interact with pathogens electrophilically, while preserving the ability of the pathogen inactivating compound to inactivate harmful pathogens. There is a need for methods of inactivating pathogens in biological materials while reducing undesired side reactions.
Methods are provided for quenching compounds comprising reactive groups in materials. A variety of compounds may be quenched in materials, such as biological materials, using the methods disclosed herein. Compounds that may be quenched include compounds that comprise a functional group that is, or which is capable of forming, and has formed, in situ, a reactive group such as an electrophilic group. For example, the functional group may be a mustard group that is capable of forming in situ a reactive group such as an electrophilic aziridine, aziridinium, thiirane or thiiraniium ion. In another embodiment, the functional group may be an epoxide.
In one embodiment, methods are provided for quenching side reactions of pathogen inactivating compounds used to inactivate pathogens in biological materials. In a particular embodiment, methods are provided for quenching undesired side reactions of a pathogen inactivating compound that includes a functional group which is, or which is capable of forming, an electrophilic group. In this embodiment, a biological material is treated with the pathogen inactivating compound and a quencher comprising a nucleophilic functional group that is capable of covalently reacting with the electrophilic group. The electrophilic group on the pathogen inactivating compound is in one preferred embodiment a cationic group. Biological materials can be treated with the pathogen inactivating compound and the quencher, for example, in vitro or ex vivo. In a preferred embodiment, the pathogen inactivating compound and the quencher are administered to a material in an effective amount for the pathogen inactivating compound to inactivate pathogens in the material while also permitting the quencher to quench undesired side reactions of the pathogen inactivating compound.
In one embodiment, a quencher may be introduced into a multiphase system, such as a two phase system. For example, the quencher may be administered to a two phase system wherein a membrane separates the first and second phases. In one embodiment, one phase is bounded by the membrane. For example, the membrane may define the exterior membrane of a pathogenic organism, the first phase may be the phase in which the pathogenic organism is contained, and the second phase may be the interior of the pathogenic organism. The membrane may be, for example the lipid coat of a virus, the first phase may be a fluid in which the virus is disposed, such as blood, and the second phase may be the interior of the virus comprising viral nucleic acids. In another embodiment, the membrane may be a cell membrane, and the membrane bounded phase may be the interior of a pathogenic unicellular organism, such as a bacterium. In this embodiment, a pathogen inactivating compound is introduced into the two phase system that preferably is kinetically or thermodynamically capable of traversing the membrane, while the quencher substantially is not kinetically or thermodynamically capable of traversing the membrane, relative to the pathogen inactivating compound.
In one embodiment, a method is provided for quenching undesired side reactions of a pathogen inactivating compound in a two phase biological material comprising a first liquid phase having a pathogen comprising a membrane therein, and a second phase bounded by the membrane of the pathogen. The second phase thus is the contents contained within the pathogen membrane. The biological material is treated with a pathogen inactivating compound comprising a functional group which is capable of forming an electrophilic group. Prior to, contemporaneously, or after treatment with the pathogen inactivating compound, the biological material is treated with a quencher comprising a nucleophilic group that is capable of covalently reacting with the electrophilic group. Preferably, the quencher is added prior to or contemporaneously with the pathogen inactivating compound. Preferably, the pathogen inactivating compound is kinetically or thermodynamically capable of traversing the membrane, prior to formation of the electrophilic group. Preferably, the rate of membrane penetration by the pathogen inactivating compound is substantially reduced after formation of the electrophilic group, relative to the pathogen inactivating compound prior to formation of the electrophilic group. Preferably, the pathogen inactivating compound is substantially not kinetically or thermodynamically capable of traversing the membrane after formation of the electrophilic group, relative to the pathogen inactivating compound prior to formation of the electrophilic group. The quencher preferably is substantially not kinetically or thermodynamically capable of traversing the membrane, relative to the pathogen inactivating compound prior to the formation of the electrophilic group. The quencher is permitted to react with the electrophilic group of the pathogen inactivating compound, and the reaction of the quencher with the electrophilic group of the pathogen inactivating compound preferably occurs substantially in the first phase. The pathogen inactivating compound comprising the electrophilic group reacts with a nucleic acid of the pathogen in the second membrane bounded phase, thereby inactivating the pathogen. The methods permit pathogen inactivation by reaction of the pathogen inactivating compound with pathogen nucleic acids within the pathogen membrane, while permitting quenching of reactive pathogen inactivating compounds, as well as reactive species formed therefrom, in the phase in which the pathogen is disposed.
The methods disclosed herein are advantageous, since in the two phase system, the quencher is administered to the first phase and substantially is not capable of traversing the exterior membrane of the pathogen, and thus is substantially not present in the second phase, i.e., the interior of the pathogen. The pathogen inactivating compound is administered, for example, to the first phase, and is capable of traversing the membrane of the pathogen, prior to formation of the electrophilic group. Upon formation of the electrophilic group in situ, the pathogen inactivating compound substantially is no longer capable of traversing the membrane. Thus, quenching selectively occurs in the first phase, while in the second phase, i.e., the interior of the pathogen, the pathogen inactivating compound reacts with nucleic acids of the pathogen without quenching. Thus, in a biological material, such as a blood product, quenching of reactive groups on the pathogen inactivating compound and degradation products thereof occurs selectively in the first phase in which the pathogen is suspended. The quenching in the first phase thus reduces unwanted side reactions of the reactive group on the pathogen inactivating compound, such as covalent modification of proteins or cell surfaces in blood. In the method, the pathogen inactivating compound and the quencher are administered in an effective amount to inactivate pathogens in the material while quenching undesired side reactions of the pathogen inactivating compound. The quencher thus has a protective effect in reducing unwanted side reactions in materials such as blood, while permitting pathogen inactivation to occur.
The methods may be used to treat biological materials comprising pathogens, such as prokaryotic and eukaryotic organisms and lipid coated viruses. In particular, the methods may be used to treat pathogens comprising membranes, such as lipid coated viruses, and bacteria that include a membrane in the form of a cell membrane. Exemplary quenchers for treating biological materials, such as blood products, having therein a pathogen comprising a membrane, include glutathione, N-acetylcysteine, cysteine, mercaptoethanesulfonate salts, or dimercaprol.
The pathogen inactivating compound may include a nucleic acid binding ligand and a functional group which is, or which is capable of forming, an electrophilic group, wherein the electrophilic group is capable of reacting with a nucleic acid to form a covalent bond with a nucleic acid. The pathogen inactivating compound also may further include a frangible linker, linking the nucleic acid binding ligand and the functional group. For example, the functional group may be a mustard group which is capable of reacting in situ to form the electrophilic group, such as an aziridinium ion. Exemplary pathogen inactivating compounds include quinacrine mustard, N-(2-chloroethyl)-N-ethyl-Nxe2x80x2-(6-chloro-2-methoxy-9-acridinyl)-1,3-propanediamine dihydrochloride, and 5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralen. Other pathogen inactivating compounds include compounds comprising an aziridine covalently linked to a polyamine moiety, as described in Budowsky et al., Vaccine Research, 5:29-39 (1996).
Quenchers which may be used may include nucleophilic functional groups such as thiols, thioacids, dithoic acids, phosphates, thiophosphates and amines. Exemplary quenchers include glutathione, N-acetylcysteine, cysteine, thiosulfate, mercaptoethanesulfonate salts, and dimercaprol. In a preferred embodiment, the quencher is glutathione.
In methods wherein a biological material, such as a blood product, is treated with a quencher and a pathogen inactivating compound, the quencher may be added to the material prior to, simultaneously with, or after the addition of the pathogen inactivating compound. Preferably, the quencher is added prior to or contemporaneously with the pathogen inactivating compound. In another preferred embodiment, the quencher is added within about 30 minutes, for example, within about 15-20 minutes, or within about 10 minutes of addition of the pathogen inactivating compound. Biological materials which may be treated include blood products, such as whole blood, red blood cells, blood plasma, or fractions thereof, and platelets. The methods may be used to produce treated blood products that are suitable for introduction into an individual. For example, the treated material may be transfused into an individual in need thereof. Optionally, the concentration of the pathogen inactivating compound and/or quencher in the material may be reduced after the treatment, and before transfusion, for example, by filtration or adsorption. A wide variety of biological materials, such as blood products, that contain, or are suspected to contain, a pathogen may be treated.
In one particular embodiment, a method is provided for quenching undesired side reactions of a pathogen inactivating compound in a material comprising red blood cells, wherein the method includes treating a material comprising red blood cells with a pathogen inactivating compound comprising a functional group which is, or which is capable of forming, an electrophilic group, and treating the material with a quencher comprising a nucleophilic group that is capable of covalently reacting with the electrophilic group. The pathogen inactivating compound in one embodiment comprises a nucleic acid binding ligand and a mustard group that is capable of reacting in situ to form an electrophilic group, such as an aziridinium ion. The material may comprise packed red blood cells. The material may comprise, for example, a blood product with a hematocrit of about 30-85%. For example, a material comprising red blood cells may be treated with a pathogen inactivating compound and a quencher, and then the treated material may be transfused into an individual in need thereof.
The concentration of the pathogen inactivating compound may be, for example, about 0.1 xcexcM to 5 mM. A concentration of pathogen inactivating compound may be provided which is sufficient to inactivate, for example, at least about 3 to 6 logs of a pathogen in the material being treated. The nucleophilic group is preferably a thiol group. The quencher in a preferred embodiment is glutathione. The concentration of glutathione can be, for example, on the order of about 0.5 to 30 mM. The material can be incubated with the pathogen inactivating compound and the quencher, for example, for at least about 1 to 48 hours.
Preferably, the quencher reduces the inactivation of a pathogen by the pathogen inactivating compound by no greater than about 3 logs in comparison to a control pathogen inactivation conducted in the absence of the quencher. In another preferred embodiment, in the method, the quencher reduces the inactivation of a viral pathogen by the pathogen inactivating compound by no greater than about 1 log in comparison to a control pathogen inactivation conducted in the absence of the quencher. Preferably, in the embodiment wherein a material comprising red blood cells is treated, red blood cell function is not substantially altered after the treatment.
In one preferred embodiment, a method is provided, wherein the method comprises treating a red blood cell containing material with an effective amount of the pathogen inactivating compound and the quencher to inactivate at least 2 logs of a pathogen, and wherein red blood cell function is not substantially altered by the treatment. In another preferred embodiment, a method is provided wherein the method comprises treating the material with an effective amount of the pathogen inactivating compound and the quencher to inactivate at least 2 logs of a pathogen, and wherein hemolysis of the red blood cells is less than 3% after 28 days of storage.